Method for producing a soy protein product

ABSTRACT

The present invention relates to a method for producing a soy protein product by treatment of soy protein with at least one oxidoreductase.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. 119 of Danishapplication no. PA 2005 01076 filed Jul. 20, 2005 and the benefit under35 U.S.C. 119 of U.S. provisional application No. 60/701,709 filed Jul.21, 2005 the contents of which are fully incorporated herein byreference.

TECHNICAL FIELD

The present invention relates to a method for producing a soy proteinproduct by treating soy protein with an oxidoreductase.

BACKGROUND OF THE INVENTION

Soy protein products, such as soy flour, soy protein concentrates andsoy protein isolates, are used as ingredients in a large number ofproducts, such as in food products. The functional properties of soyprotein products are important for their quality as ingredients.Important functional properties are properties like water binding,ability to impart textural properties, flavour and taste. There is aneed for soy protein products with improved functional properties, e.g.improved ability to impart textural properties such as viscosity to foodproducts. Enzymatic processing of soy protein is known in the art. WO97/43910 discloses a method for producing soy protein hydrolysates withexcellent flavour by subjecting the soy protein to deamidation, forexample enzymatic deamidation, and subjecting the soy protein to aspecific acting proteolytic enzyme.

SUMMARY OF THE INVENTION

The inventors have found that treating soy protein with at least oneoxidoreductase results in a soy protein product with improved functionalproperties. Accordingly the present invention relates to a method forproducing a soy protein product comprising treating soy protein with anoxidoreductase.

DETAILED DISCLOSURE OF THE INVENTION

Oxidoreductases

An oxidoreductase may be any oxidoreductase described by the enzymeclassification EC 1 as set out by the Nomenclature Committee of theInternational Union of Biochemistry and Molecular Biology (IUBMB), orany fragment derived therefrom exhibiting oxidoreductase activity. Inone embodiment of the invention, an oxidoreductase is an oxidoreductaseacting on diphenols and related substances as donors comprised by theenzyme classification EC 1.10, such as a laccase (EC 1.10.3.2), ano-aminophenol oxidase (EC 1.10.3.4), or a catechol oxidase (EC1.10.3.1); or an oxidoreductase acting on CH—OH groups of donorsdescribed by the enzyme classification EC 1.1, such as a peroxidase, aglucose oxidase (EC 1.1.3.4), a hexose oxidase (EC 1.1.3.5), or acellobiose oxidase (EC 1.1.3.25). In one embodiment of the invention,the soy protein is treated with a combination of two or moreoxidoreductases, e.g. a combination of a peroxidase and a glucoseoxidase (EC 1.1.3.4), a hexose oxidase (EC 1.1.3.5), or a cellobioseoxidase (EC 1.1.3.25). In a further embodiment of the invention, theoxidoreductase is a lipoxygenase (EC 1.13.11.12).

An oxidoreductase may be of any origin, e.g. of microbial origin. Theenzyme may e.g. be derived from animals, plants, bacteria or fungi(including filamentous fungi and yeasts).

Suitable examples of fungal laccases include laccases derivable from astrain of Aspergillus, Neurospora, e.g., N. crassa, Podospora, Botrytis,Collybia, Fomes, Lentinus, Pleurotus, Trametes, e.g., T. villosa and T.versicolor, Rhizoctonia, e.g., R. solani, Coprinus, e.g., C. cinereus,C. comatus, C. friesii, and C. plicatilis, Psathyrella, e.g., P.condelleana, Panaeolus, e.g., P. papilionaceus, Myceliophthora, e.g., M.thermophila, Schytalidium, e.g., S. thermophilum, Polyporus, e.g., P.pinsitus, Phlebia, e.g., P. radita (WO 92/01046), or Coriolus, e.g., C.hirsutus (JP 2-238885).

Suitable examples of laccases from bacteria include a laccase derivablefrom a strain of Bacillus.

The oxidoreductase may furthermore be one which is producible by amethod comprising cultivating a host cell transformed with a recombinantDNA vector which carries a DNA sequence encoding said oxidoreductase aswell as DNA sequences encoding functions permitting the expression ofthe DNA sequence encoding the oxidoreductase, in a culture medium underconditions permitting the expression of the oxidoreductase enzyme, andrecovering the oxidoreductase from the culture.

Determination of Laccase Activity (LACU)

Laccase activity (particularly suitable for Polyporus laccases) may bedetermined from the oxidation of syringaldazin under aerobic conditions.The violet colour produced is measured with a spectrophotometer at 530nm. The analytical conditions are 19 mM syringaldazin, 23 mM acetatebuffer, pH 5.5, 30° C., 1 min. reaction time.

1 laccase unit (LACU) is the amount of enzyme that catalyses theconversion of 1.0 micromole syringaldazin per minute at theseconditions.

Determination of Laccase Activity (LAMU)

Laccase activity may be determined from the oxidation of syringaldazinunder aerobic conditions. The violet colour produced is measured at 530nm. The analytical conditions are 19 mM syringaldazin, 23 mMTris/maleate buffer, pH 7.5, 30° C., 1 min. reaction time.

1 laccase unit (LAMU) is the amount of enzyme that catalyses theconversion of 1.0 micromole syringaldazin per minute at theseconditions.

Source of Oxygen

The source of oxygen required by the oxidoreductase may be oxygen fromthe atmosphere or an oxygen precursor for in situ production of oxygen.Oxygen from the atmosphere will usually be present in sufficientquantity. If more O₂ is needed, additional oxygen may be added, e.g. aspressurized atmospheric air or as pure pressurized O₂.

Soy Protein

Soybeans belong to the legume family and contain on average 35-40%protein. Soy protein is made from dehulled, defatted soybean meal. Theconcentration of protein is achieved by removing most of the solublenon-protein compounds. These compounds are mainly soluble carbohydratesand some nitrogenous substances and minerals. This process removes muchof the undesirable beany flavour as well as oligosaccharides (raffinoseand stachyose). It is further contemplated that the whole soybeans usedin the process of the present invention may be standard, commoditizedsoybeans, soybeans that have been genetically modified (GM) in somemanner, or non-GM identity preserved soybeans.

The term “soy protein” typically refers to processed, edible, drysoybean products other than animal feed meals. Many types are producedfor use in human and pet foods, milk replacers, and starter feeds foryoung animals. Soybean protein materials which are useful within thepresent invention are soy protein flour, soy protein concentrate, andsoy protein isolate, or mixtures thereof.

The traditional processes for making the soy protein materials includingsoy protein flours, soy protein concentrates, and soy protein isolatesall begin with the same initial steps. Soybeans entering a processingplant must be sound, mature, yellow soybeans. The soybeans can be washedto remove dirt and small stones. They are typically screened to removedamaged beans and foreign materials, and may be sorted to uniform size.

Each cleaned, raw soybean is then cracked into several pieces, typicallysix(6) to eight (8), to produce soy chips and hulls. The hulls areremoved by aspiration. Alternatively, the hulls may be loosened byadjusting the moisture level and mildly heating the soybeans beforecracking. Hulls can also be removed by passing cracked pieces throughcorrugated rolls revolving at different speeds. In these methods, thehulls are then removed by a combination of shaker screens andaspiration.

Soy chips, which contain about 11% moisture, are then conditioned atabout 60° C. and flaked to about 0.25 millimeter thickness. Theresulting flakes are then extracted with an inert solvent, such as ahydrocarbon solvent, typically hexane, in one of several types ofcountercurrent extraction systems to remove the soybean oil. Hexaneextraction is basically an anhydrous process, as with a moisture contentof only about 11%, there is very little water present in the soybeans toreact with the protein. For soy protein flours, soy proteinconcentrates, and soy protein isolates, it is important that the flakesbe desolventized in a manner which minimizes the amount of cooking ortoasting of the soy protein to preserve a high content of water-solublesoy protein. This is typically accomplished by using vapourdesolventizers or flash desolventizers. The flakes resulting from thisprocess are generally referred to as “edible defatted flakes.” Speciallydesigned extractors with self-cleaning, no-flake-breakage features, andthe use of a narrow boiling range hexane are recommended for producingedible defatted flakes.

The resulting edible defatted flakes, which are the starting materialfor soy protein flour, soy protein concentrate, and soy protein isolate,have a protein content of approximately 50%. Moisture content hastypically been reduced by three (3) to five (5)% during this process.Any residual solvent may be removed by heat and vacuum.

The soy protein flour, soy protein concentrate, and soy protein isolateare described below as containing a protein range based upon a “moisturefree basis” (mfb).

The edible defatted flakes are then milled, usually in an open-loopgrinding system, by a hammer mill, classifier mill, roller mill orimpact pin mill first into grits, and with additional grinding, into soyflours with desired particle sizes. Screening is typically used to sizethe product to uniform particle size ranges, and can be accomplishedwith shaker screens or cylindrical centrifugal screeners.

Soy Protein Flour

Soy protein flour, as that term is used herein, refers to a comminutedform of defatted soybean material, preferably containing less than 1%oil and formed of particles having a size such that the particles canpass through a No. 100 mesh (U.S. Standard) screen. Soy protein flourhas a soy protein content of about 50% to about 65% on a moisture freebasis (mfb). Preferably the flour is very finely ground, most preferablyso that less than about 1% of the flour is retained on a 300 mesh (U.S.Standard) screen. The remaining components are soy fiber material, fats,minerals, and sugars such as sucrose, raffinose and stachyose.

Soy Protein Concentrate

Soy protein concentrate, as the term is used herein, refers to a soyprotein material containing from about 65% to less than about 90% of soyprotein (mfb). The remaining components are soy fiber material, fats,minerals, and sugars such as sucrose, raffinose, and stachyose. Soyprotein concentrates are prepared from dehulled and defatted soy flakesby removing most of the water-soluble, non-protein constituents. The“traditional method” for preparing soy protein concentrates is byaqueous alcohol leaching. In this method, edible defatted soy flakes areleached (washed) with alcohol and water. The alcohol and water istypically 60% to 90% ethanol, and removes much of the soluble sugars.The soluble sugars are separated from the wet flakes with the solublesugars being used for some other purpose or discarded. The wet flakesare transferred to a desolventizer. Sufficient heat is used in thedesolventizer to increase the vapor pressure of the alcohol and water toremove that liquid, but is sufficiently low enough to minimize cookingof the protein. The application of reduced pressures over the liquidbearing mass also increases the rate of removal of the liquid.

The remaining water and wet flakes are dried in a dryer to remove waterand to produce a soy protein concentrate.

Secondary treatments such as high pressure homogenization or jet cookingcan be used to restore some solubility lost during processing.

Another less used method for producing soy protein concentrates is byacid leaching. Edible defatted flakes and water are combined in a ratioof about 10:1 to 20:1 water to edible defatted flakes, with a food-gradeacid (water plus acid) typically hydrochloric acid, to adjust the pH toabout 4.5. The extraction typically runs for about 30 to 45 minutes atabout 40° C. The acid-leached flakes are separated from the acidsolubles to concentrate the solids to about 20%. A second leach andcentrifugation may also be employed. The acid solubles are used for someother purpose or are discarded. The acidified wet flakes are neutralizedto a pH of about 7.0 with alkali and water (e.g., sodium hydroxide orcalcium hydroxide) to produce neutralized water and wet flakes. Theneutralized water is separated from the wet flakes and the wet flakesare spray dried at about 157° C. inlet air temperature and about 86° C.outlet temperature to remove water and to produce soy proteinconcentrate. Soy protein concentrates are commercially available fromSolae® LLC, for example, as Promine DSPC, Procon, Alpha 12 and Alpha5800.

Soy protein concentrates are available in different forms, e.g. asgranules or spray dried product.

Soy Protein Isolate

Soy protein isolate, as the term is used herein, refers to a soy proteinmaterial containing at least about 90% protein content (mfb). Theremaining components are soy fiber material, fats, minerals, and sugarssuch as sucrose, raffinose, and stachyose. The edible defatted flakesare placed in an aqueous bath to provide a mixture having a pH of atleast about 6.5 and preferably between about 7.0 and about 10.0 in orderto extract the protein. Typically, if it is desired to elevate the pHabove 6.7, various alkaline reagents such as sodium hydroxide, potassiumhydroxide and calcium hydroxide or other commonly accepted food gradealkaline reagents may be employed to elevate the pH. A pH of above about7.0 is generally preferred, since an alkaline extraction facilitatessolubilization of the soy protein. Typically, the pH of the aqueousextract of soy protein will be at least about 6.5 and preferably about7.0 to about 10.0. The ratio by weight of the aqueous extractant to theedible defatted flakes is usually between about 20:1 and preferably aratio of about 10:1. Before continuing a work-up of the extract, theextract is centrifuged to remove insoluble carbohydrates. A secondextraction is performed on the insoluble carbohydrates to remove anyadditional soy protein. The second extract is centrifuged to give anyfurther insoluble carbohydrates and a second aqueous extract. The firstand second extracts are combined for the work-up. The insolublecarbohydrates are used to obtain the soy fiber. In an alternativeembodiment, the soy protein is extracted from the edible defatted flakeswith water, that is, without a pH adjustment.

The extraction temperatures which may be employed can range from ambientup to about 49° C. (120° F.) with a preferred temperature of 32.2° C.(90° F.). The period of extraction is further non-limiting and a periodof time between about 5 to about 120 minutes may be convenientlyemployed with a preferred time of about 30 minutes. Following extractionof the soy protein material, the aqueous extract of soy protein can bestored in a holding tank or suitable container while a second extractionis performed on the insoluble solids from the first aqueous extractionstep. This improves the efficiency and yield of the extraction processby exhaustively extracting the soy protein from the residual solids fromthe first step.

The combined, aqueous soy protein extracts from both extraction steps,without the pH adjustment or having a pH of at least 6.5, or preferablyabout 7.0 to about 10, are then precipitated by adjustment of the pH ofthe extracts to, at or near the isoelectric point of the soy protein toform an insoluble curd precipitate. The pH to which the soy proteinextracts are adjusted is typically between about 4.0 and about 5.0. Theprecipitation step may be conveniently carried out by the addition of acommon food grade acidic reagent such as acetic acid, sulfuric acid,phosphoric acid, hydrochloric acid, or with any other suitable acidicreagent. The soy protein precipitates from the acidified extract, and isthen separated from the extract. The separated soy protein may be washedwith water to remove residual soluble carbohydrates and ash from theprotein material and the residual acid can be neutralized to a pH offrom about 4.0 to about 6.0 by the addition of a basic reagent such assodium hydroxide or potassium hydroxide. At this point the soy proteinmaterial is subjected to a pasteurization step. The pasteurization stepkills microorganisms that may be present. Pasteurization is carried outat a temperature of at least 82.2° C. (180° F.) for at least 10 seconds,at a temperature of at least 87.8° C. (190° F.) for at least 30 secondsor at a temperature of at least 90.6° C. (195° F.) for at least 60seconds. The soy protein material is then dried using conventionaldrying means to form a soy protein isolate. Soy protein isolates arecommercially available from Solae® LLC, for example, as SUPRO® 500E,SUPRO® PLUS 651, SUPRO® PLUS 675, SUPRO® 516, SUPRO® XT 40, SUPRO® 710,SUPRO® 720, FXP 950, FXP H0120 and PROPLUS 500F.

The soy protein material used in the present invention, may be modifiedto enhance the characteristics of the soy protein material. Themodifications are modifications which are known in the art to improvethe utility or characteristics of a protein material and include, butare not limited to, denaturation and hydrolysis of the protein material.

Treatment with Oxidoreductase

The soy protein to be treated may be in an aqueous suspension. Anaqueous suspension may be prepared by mixing a soy protein preparationwith water. Additional ingredients, such as salts, may be addeddepending on the desired properties of the final product. The pH andionic strength of the aqueous suspension may be controlled to providesuitable conditions for the oxidoreductase enzyme to be active,depending on the desired properties of the final soy protein product. Inone embodiment of the invention, an aqueous preparation of soy proteincontaining at least 50%, preferably at least 60%, more preferably atleast 70%, and more preferably at least 80%, soy protein (weight/weight)in dry matter is treated with an oxidoreductase. In another embodiment,an aqueous suspension of soy protein concentrate and/or soy proteinisolate is treated with an oxidoreductase.

The treatment with oxidoreductase may be effected by adding theoxidoreductase, as a dry product, a suspension, or a solution to anaqueous solution or suspension of soy protein. The oxidoreductase may bemixed into the solution or suspension of soy protein by any appropriatemeans known in the art. Additionally a mediator may be added. A mediatormay be any substance suitable for enhancing the action of theoxidoreductase on the soy protein, such as tyrosine.

Treatment of soy protein with an oxidoreductase according to theinvention may be performed in the presence of carbohydrates, lipids,hydrogen peroxide, other proteins, and mixtures thereof.

The temperature of the oxidoreductase treatment may be any temperaturesuitable for ensuring the activity of the specific oxidoreductase enzymeused. Typically, the range is between 5° C. and 100° C. The temperaturemay be chosen by the skilled person by methods well known in the art. Inone embodiment of the invention, soy protein is treated with anoxidoreductase at a temperature between 5° C. and 100° C., preferablybetween 10° C. and 80° C., and more preferably between 50° C. and 70° C.Similarly the treatment of soy protein with an oxidoreductase may beperformed at a pH chosen by methods known in the art depending on thespecific enzyme and/or soy protein material. In one embodiment of theinvention, soy protein is treated with an oxidoreductase at a pH between2 and 10, preferably between 4 and 9, more preferably between 6 and 8.The duration of the treatment may be any duration suitable for obtainingthe desired result. Typically, the duration of the treatment of soyprotein with oxidoreductase is between 5 minutes and 5 hours. In oneembodiment, the duration of the treatment of soy protein with anoxidoreductase is between 5 minutes and 5 hours, preferably between 10minutes and 2 hours. The amount of oxidoreductase enzyme used may bechosen so as to achieve the desired result. Typically, the amount ofoxidoreductase enzyme used is between 0.5 LAMU/g soy product and 4.0LAMU/g soy product. The amount depends on the activity of the specificoxidoreductase towards the specific soy protein substrate, along withthe temperature, duration, and other conditions of the oxidoreductasetreatment.

In another embodiment of the invention, soy protein is treated with anamount of oxidoreductase and for a time sufficient to lead to anincrease in water holding capacity of the treated soy proteinpreparation compared to similar untreated soy protein. Typically the soyprotein is treated with between 0.5 LAMU/g soy product and 4.0 LAMU/gsoy product of oxidoreductase for at least 30 minutes at a temperatureof between 40° C. and 70° C., in order to increase the water holdingcapacity of the treated soy protein preparation.

In one embodiment the invention relates to use of at least oneoxidoreductase to treat soy protein concentrate and/or soy proteinisolate to increase the water holding capacity and/or water binding ofthe soy protein concentrate and/or soy protein isolate. In a furtherembodiment the invention relates to use of at least one oxidoreductaseto treat soy protein concentrate and/or soy protein isolate to increasethe viscosity of a solution or suspension of the soy protein concentrateand/or soy protein isolate.

Functional Properties of Soy Protein Preparation of the Invention

The soy protein preparation of the invention has improved propertiescompared to a similar soy protein product that has not been treated withan oxidoreductase. In one embodiment, the viscosity of a suspension ofthe soy protein preparation is higher than a similar suspension that hasnot been treated with an oxidoreductase. Typically the soy protein istreated with between 0.5 LAMU/g soy product and 4.0 LAMU/g soy productof oxidoreductase for at least 30 minutes at a temperature of between40° C. and 70° C., in order to increase the viscocity of the treated soyprotein preparation. Viscosity may be increased by at least 2%,preferably at least 3%, and more preferably at least 5%, compared to asimilar soy protein product that has not been treated with anoxidoreductase. Viscosity may be measured by methods well known in theart, e.g. by rotary viscometry. In another embodiment, the taste of thesoy protein preparation is improved. In still another embodiment, thewater holding capacity of the soy protein preparation is increasedcompared to a similar soy protein preparation that has not been treatedwith an oxidoreductase. Inclusion of a soy protein preparation accordingto the invention in a food product may increase the water holdingcapacity of the food product compared to a similar food productcomprising an equal amount of a similar soy protein preparation producedwithout treatment with an oxidoreductase. Water holding capacity is theability of a material to hold its own and/or added water during theapplication of forces, pressing, and/or heating. Water holding capacitymay be evaluated by measurement of viscosity, by the method furnished byAACC (American Association of Cereal Chemists) Technical Committee, asdescribed in Cereal Foods World (1981) 26:291, and/or by the methoddescribed in the examples following hereafter. In one embodiment of theinvention, the water binding of the soy protein product is improved bythe treatment with an oxidoreductase. The amount of bound water may bedetermined by ¹HNMR as described by H. C. Bertram et al., J. Agric. FoodChem., 50, 824-829 (2002) and in the examples following hereafter.

Treatment of soy protein with oxidoreductase may lead to crosslinking ofsoy protein molecules, e.g. by formation of dityrosine.

Food Product

In one embodiment, the invention relates to a method for producing afood product comprising mixing a soy protein preparation of theinvention with additional food ingredients and producing a food productfrom the mixture. A food product of the invention may be any foodproduct, e.g. a meat product, a dairy product, a vegetable product,fruit product, a ready to eat product, and mixtures thereof. A foodproduct of the invention may also be a component of a food used toimpart desired form or structure, enhance texture, or improveconvenience in use, e.g. an edible film, coating or casing.

Soy protein is well known in the art as an ingredient of or an additiveto a number of different food products. A soy protein preparationaccording to the invention may be used as an ingredient in a foodproduct in the same way as other soy protein products are usually used.A food product comprising a soy protein preparation according to theinvention may be produced in the same manner as a food productcomprising a conventional soy protein product. A soy protein preparationaccording to the invention may be added in the same way and in the sameamounts as a conventional soy protein product is added to a similar foodproduct.

A meat product according to the invention may be a whole meat product ora processed meat product, such as sausage, meat loaf, comminuted meatproduct, ground meat, bacon, polony, salami, or pate. A processed meatproduct may further comprise salts, spices, milk protein, vegetableingredients, colouring agents, texturising agents, and mixtures thereof.A processed meat product may be an emulsified meat product, manufacturedfrom a meat based emulsion. The meat based emulsion may be cooked orbaked in a baking form or after being filled into casing of plastic,collagen, cellulose, or natural casing. A processed meat product mayalso be a restructured meat product, such as restructured ham. A meatproduct of the invention may undergo at least one of the followingprocessing steps: curing, drying, smoking, fermentation, cooking,slicing, and/or shredding. A meat based food product may be produced bycontacting meat with a soy protein preparation according to theinvention and producing a meat based food product from the treated meat.The meat will usually be raw when being contacted with a soy proteinpreparation according to the invention, but may also be heat treated,precooked, or irradiated. The meat may also have been frozen beforecontact with a soy protein preparation according to the invention.Contacting meat with a soy protein preparation according to theinvention may be done by adding a soy protein preparation according tothe invention to meat. Contacting meat with a soy protein preparationaccording to the invention may be achieved by mixing meat, such aspieces of meat, minced meat, or a meat based emulsion, with a soyprotein preparation according to the invention and, where applicable,other ingredients used to form the meat based food product by any methodknown in the art. Before contact with the meat, a soy proteinpreparation according to the invention may be mixed with otheringredients, to form a marinade or pickling liquid, such as water, salt,flour, milk protein, vegetable protein, starch, hydrolysed protein,phosphate, acid, spices, and mixtures thereof. The amount of a soyprotein preparation according to the invention in a marinade may beadjusted as to achieve the desired final amount of a soy proteinpreparation according to the invention in the meat based food product.Contacting meat, such as whole animal muscle or pieces of animal muscle,with a soy protein preparation according to the invention may beachieved by marinating and/or tumbling and/or injecting the meat with amarinade comprising a soy protein preparation according to theinvention. If the meat product is a processed meat product, such as anemulsified meat product, a soy protein preparation according to theinvention may be mixed into a meat based emulsion, or into any otherform of meat based mixture used to form the processed meat product.

A dairy product according to the invention may be skimmed milk, wholemilk, cream, a fermented milk product, cheese, yoghurt, butter, dairyspread, butter milk, acidified milk drink, sour cream, whey based milkdrink, ice cream, a flavoured milk drink, or a dessert product based onmilk components such as vla or custard. A dairy product may additionallycomprise non-milk components, such as vegetable components includingvegetable oil, vegetable protein, vegetable carbohydrates, and mixturesthereof. Dairy products may also comprise further additives such asenzymes, flavouring agents, microbial cultures, salts, sweeteners,sugars, acids, fruit, fruit juices, any other component known in the artas a component of, or additive to a dairy product, and mixtures thereof.

EXAMPLE 1

Materials

Soy protein concentrate A

Soy protein concentrate B

Soy protein isolate

Laccase (derived from a strain of Myceliophthora thermophila asdisclosed in WO 95/33836)

Soy Samples

Three separate samples were run in which the 6% soy protein was Soyprotein concentrate A in one sample, Soy protein concentrate B in asecond sample, and Soy protein isolate in a third sample. Results appearin Table 1 below.

The 6% soy protein was suspended in water and allowed to hydrate at 5°C. over night and then treated with 0-0.5 LAMU/g laccase for 30 minutesat 60° C. (6° dH, pH 7.3) and subsequently heat treated for 10 minutesat 95° C.

Viscosity Measurement:

Viscosity was measured on a Rapid Visco Analyzer, model RVAA at 200 rpmfor 2 minutes at 23° C. Triple determinations were made with 25 g ofeach sample suspension.

Precipitate Determination:

Centrifugation was carried out as follows: 10 min. at 3500 rpm, 20 ° C.in a Heraeus Sorvall multifuge 3 S-R. Amount of precipitate wasdetermined by decanting and weighing the precipitate.

Results TABLE 1 Laccase Average LAMU/g soy viscosity Precipitate Soysample protein cP G Soy protein concentrate A 0 93 Soy proteinconcentrate A 0.1 107 Soy protein concentrate A 0.25 103 Soy proteinconcentrate A 0.5 107 Soy protein concentrate B 0 57 3.61 Soy proteinconcentrate B 0.1 60 3.95 Soy protein concentrate B 0.25 60 3.85 Soyprotein concentrate B 0.5 61 3.93 Soy protein isolate 0 53 0.88 Soyprotein isolate 0.1 53 1.02 Soy protein isolate 0.25 54 1.08 Soy proteinisolate 0.5 57 1.28

EXAMPLE 2

Materials:

Soy protein isolate (Supro® 500E, The Solae® Company)

Soy protein concentrate (Alpha 12TS, The Solae® Company)

Soy Flour (The Solae® Company)

8 g of soy product was dissolved in 92 g of distilled water by slowaddition under magnetic stirring. Laccase was added to the dissolved soyproduct under magnetic stirring at 60° C. at a dose of 1.0 LAMU/g soyproduct, and samples were left for 30 minutes for enzymatic reaction andthen heated to 90° C. for 10 minutes in a shaking water bath toinactivate the laccase. Control samples were treated in the same wayexcept that no laccase was added.

Viscosity of the treated samples was measured as described in Example 1.Results are shown in Table 2. TABLE 2 Viscosity (cP) Soy product Controlsample Laccase treated sample Soy protein isolate 72 101 Soy proteinconcentrate 101 112 White flakes 38 41

EXAMPLE 3

Materials:

Soy protein isolate (Supro® 500E, The Solae® Company)

Soy protein concentrate (Alpha 12TS, The Solae® Company)

Soy Flour (The Solae® Company)

4 g of soy product was dissolved in 59 g of distilled water by slowaddition under magnetic stirring. Laccase was added to the dissolved soyproduct under magnetic stirring at 60° C. at a dose of 1.5 LAMU/g soyproduct, and samples were left for 30 minutes for enzymatic reaction andthen heated to 90° C. for 5 minutes in a shaking water bath toinactivate the laccase. Control samples were treated in the same wayexcept that no laccase was added.

Model sausages were produced by mixing 35 g of minced pork meat withmaximum 12% fat with 2 g salt and 63 g of soy product solution at atemperature below 10° C. in a food processor. 4 g of the mixture wastransferred to a NMR glass tube and heat treated at 90° C. for 5 minutesin a shaking water bath.

The amount of bound and free water was determined by low resolution¹HNMR as described by H. C. Bertram et al., J. Agric. Food Chem., 50,824-829 (2002). Relaxation time measurements were performed on a MaranUltra ¹H 24 MHz (Resonance Instruments, UK) with 4095 echoes, TAU of 150microseconds and relaxation delay of 4 seconds. The amount of boundwater was determined as the area of the signal component denoted T21 byBertram et. al with a relaxation time around 45 ms. Relaxation timeswere observed between 80 and 90 ms for soy protein concentrate and soyprotein isolate and between 75 and 95 ms for white flakes, for the samecomponent. The total amount of water was determined as the sum of theareas of all components.

The amount of bound water as percent of total water is shown in Table 3.TABLE 3 Amount of bound water in percent of total water Soy productControl sample Laccase treated sample Soy protein isolate 53% 55% Soyprotein concentrate 62% 70% White flakes 76% 74%

EXAMPLE 4

Materials:

Soy protein concentrate (Alpha 12TS, The Solae® Company)

Model sausages were prepared as described in Example 3 with soy proteinconcentrate treated with varying amounts of laccase (0.5, 1.0, 1.5 and4.0 LAMU/g soy protein concentrate) and the amount of bound waterdetermined as described in example 3. The increase in amount of boundwater compared to control sausages prepared with soy protein concentratethat was not treated with laccase is given in Table 4. TABLE 4 Increasein amount of bound water compared to untreated control. LAMU Laccase pr.g soy Increase in amount of bound protein concentrate water compared tocontrol 0.5 12% 1.0 17% 1.5  6% 4.0  0%

EXAMPLE 5

Materials:

Soy protein concentrate

Samples were produced by dissolving soy protein concentrate to 12.50%Total Solids. Sodium hydroxide was used to adjust the pH to 7.2. Afterneutralization, the slurry was heated to 153.3° C. (308° F.) and held atthis temperature for 15 seconds and then cooled to 54° C. (129° F.).

Laccase was added in various amounts (0.435, 0.869 and 1.736 LAMU/g soyprotein concentrate) under stirring at a temperature of 50° C. (122° F.)and allowed to react at this temperature for 30 minutes. After theenzyme reaction the sample was heated to 151.7° C. (305° F.) for 10-15seconds. The sample was spray dried and the product was collected foruse and analysis. Control samples were prepared in the same way exceptthat no enzyme was added.

Detection of Dityrosine

Dityrosine is a well characterized protein oxidation product frombiological systems, it is acid stable and can be detected upon acidhydrolysis by HPLC separation followed by fluorescence detection.Dityrosine was measured as a measure of the degree of cross-binding as aresult of the laccase treatment.

50 mg freeze dried samples were mixed with 2 mL 6 M HCl, flushed withnitrogen and hydrolyzed over night at 105° C. Subsequently, the samplesneutralized by 6 M NaOH and filtered through 0.45 micrometer filters.Twenty microliters of the hydrolyzed sample was injected onto a HPLCcolumn (Nucleosil 120-5, C-18, 250×4 mm, Macherey-Nagel, Duren, Dûren,Germany), which was equilibrated with 4% acetonitrile in aqueous 0.1 Mcitric acid (pH 2.55) with a flow of 1 mL/min as described by Daneshvaret al. (Daneshvar, B; Frandsen, H.; Dragsted, L. O.; Knudsen, L. E. andAutrup, H. (1997). Analysis of native human plasma proteins andhaemoglobin for the presence of bityrosine by high-performance liquidchromatography. Pharmacol. Toxicol. 81, 205-208). Chromatographicseparation was performed on a Varian 9012 HPLC pump, connected to aVarian 9100 Auto sampler and a Varian 9075 Fluorescence detector (VarianChromatographic Systems, Walnut Creek, Calif.). Dityrosine wasquantified using a standard curve made by the use of a dityrosinestandard prepared according to Nomura et al. (Nomura, K.; Suzuki, N. andShigenibou, M. (1990). Pulcherosine, a novel tyrosine-derived trivalentcross-linking amino acid from the fertilization envelope of sea urchinembryo. Biochemistry, 29, 4525-4534).

Water Holding Capacity of Sausages

Sausages were prepared with the treated soy protein concentrate from thefollowing ingredients: Pork Trim 80/20 16.80% Pork Back Fat 05/95 15.66%Skin Emulsion 10.00% Chicken MDM 20.00% Ice/Water 28.66% Soy ProteinConcentrate 4.00% Potato Starch 2.00% Salt 1.70% Spices 1.18%

Pork trim and pork back fat were ground before use and all meat wasstored at 4° C. before use. All ingredients were mixed and chopped for1-3 minutes and filled into cellulose casings. The meat was equilibratedfor 10 min. at 35.6° C. (96° F.), smothered for 15 min total at 43.3° C.(110° F.) with steam and heated to 65.6° C. (150° F.) (dry heat).Sausages were cooked without smoking for a total of 20 min at 60° C.(140° F.) with steam, and then at 77.8° C. (172° F.) (dry heat).

Calculation of Water Holding Capacity (WHC)

The application samples were subjected to analysis using a TA-HDITexture Analyzer from Texture Technologies Corporation, Scarsdale, N.Y.The texture analyzer generated a graph of the force used for compressionas a function of time. Samples were compressed twice to generate twopeaks in the graph. The program on the Texture Analyzer that is run forWHC is a 2-cycle TPA (Texture Profile Analysis). The test parameters are4 inch diameter flat plate probe, 2 millimeters per second probe speedhaving a 75% compression.

Hardness was determined as the maximum force during the firstcompression.

The following parameters were defined according to Bourne M. C. 1978,Texture Profile Analysis. Food Technology, 32 (7), 62-66, 72.:

Chewiness=(Gumminess×Springiness).

Gumminess=(Hardness×Cohesiveness).

Cohesiveness=(Area under second peak/Area under first peak)

Springiness=(time from beginning of second peak to the top of secondpeak/time from beginning of first peak to top of first peak)

Hardness and Chewiness were determined for each sample at hot and coldtemperatures. The cold temperature was defined as room temperature, andhot temperature was achieved by soaking of the samples in 100° C. waterbath for 34 minutes.

For two standard samples, Cold Hardness data was plotted on the y-axisand assigned WHC on the x-axis. The standards was assigned WHC of 5.0and 6.5, respectively (arbitrary units). These two points are connectedby a curve of the equation y=m×n, where y is hardness, x is WHC, and mand n are found by regression This equation was then used to calculateWHC based on cold hardness for all the samples. This type of calculationwas repeated to obtain WHC from Hot Hardness, Cold Chewiness, and HotChewiness data. These four WHC values were then averaged for each sampleto determine the Overall Water Holding Capacity. Relative comparisonscan be made between WHC values for experimental samples and appropriatecontrols.

Results

The amount of dityrosine of treated soy protein concentrate and controlas well as the increase in Water Holding Capacity of sausages producedwith the soy protein concentrate as compared to control sausages(produced with soy protein concentrate not treated with laccase) isshown in Table 5. TABLE 5 Amount of dityrosine of treated soy proteinconcentrate and control samples; and increase in Water Holding Capacityof sausages produced with the soy protein concentrate as compared tocontrol sausages (produced with soy protein concentrate not treated withlaccase). Increase in Water Laccase Dityrosine Holding Capacity comparedLAMU/g substrate (mmol/g) to control (%) 0 (Control) 25 0 0.435 35 5.80.869 37 7.7 1.736 40 16.8

EXAMPLE 6

Samples were produced by dissolving soy protein concentrate to 12% TotalSolids. Sodium hydroxide was used to adjust the pH to 7.1. Afterneutralization, the slurry was heated to 50° C. (122° F.) and laccasewas added in an amount of 0.75 LAMU/g soy protein under stirring at atemperature of 50° C. (122° F.) and allowed to react at this temperaturefor 30 minutes. After the enzyme reaction the sample was heated to151.7° C. (305° F.) for 10-15 seconds. The sample was spray dried andthe product was collected for use and analysis. Control samples wereprepared in the same way except that inactivated enzyme was added.

Determination of Molecular Weight Distribution

The molecular weight distribution was determined by size exclusionchromatography using a Supelc® TSK gel 300×7.8 mm column followed by aMicra-Synchopak SPCG-PEP 30 300×7.8 mm column mounted on aHewlett-Packard Series 1050 HPLC system equipped with a UV/Vis detectoroperating at 260 and 280 nm. The mobile phase was a phosphate buffercontaining 6 M GuanidineHCL with pH 7.6. Samples were dissolved in themobile phase before injection. As molecular weight standards were used:Hexanal, DNA, alpha-chymotrypsin, Bovines serum albumin, myoglobulin,aprotin, ovalbumin and cytochrome C. Table 6 shows the absorption at 280nm as a function of the molecular weight corresponding to the elutiontime.

Water Holding Capacity was determined as described in example 5. Thelaccase treated sample had 12% higher water holding capacity than thecontrol sample. TABLE 6 Molecular weight distribution determined by sizeexclusion HPLC Molecular weight distribution determined Molecular fromabsorbance at 280 nm (%) Weight (Da) Control Laccase treated 85936-100000* 11.9 12.2 69120-85936 12.7 13.3 52304-69120 11.0 11.734185-52304 8.6 9.3 20409-34185 6.2 6.9 12279-20409 5.2 6.0  7970-122793.4 3.9 7239-7970 2.6 3.2 6895-7239 2.0 2.7 6538-6895 1.9 2.9 5541-65382.7 3.4 4544-5541 4.8 4.1 3547-4544 6.9 5.0 1831-3547 7.0 5.0  450-18316.7 5.0 <450 6.4 5.4 Average Molecular weight: 24,370 37,399*estimated upper value

1. A method for producing a soy protein product comprising treating soyprotein with at least one oxidoreductase.
 2. The method of claim 1comprising adding oxidoreductase to a solution or suspension of soyprotein.
 3. The method of claim 1 comprising treating soy flour with atleast one oxidoreductase.
 4. The method of claim 1 comprising treatingsoy protein isolate with at least one oxidoreductase.
 5. The method ofclaim 1 comprising treating soy protein concentrate with at least oneoxidoreductase.
 6. The method of claim 1 comprising addingoxidoreductase to a solution or suspension of soy protein containing atleast 50% (weight/weight) soy protein in dry matter.
 7. The method ofclaim 1 wherein at least one oxidoreductase is added in an amountsufficient to increase the viscosity of a solution or suspension of thesoy protein.
 8. The method of claim 1 wherein at least oneoxidoreductase is added in an amount sufficient to increase the waterholding capacity and/or water binding of a solution or suspension of thesoy protein.
 9. The method of claim 1 further comprising heating thetreated soy protein to a temperature and for a time sufficient toinactivate the oxidoreductase.
 10. The method of claim 1 wherein theoxidoreductase is a laccase.
 11. The method of claim 1 furthercomprising preparing a food product with the treated soy protein.
 12. Asoy protein product produced by the method of claim
 1. 13-14. (canceled)15. The method of claim 11 wherein the food product is selected from thegroup consisting of a meat product, a dairy product, a vegetableproduct, a fruit product, a ready to eat product, and mixtures thereof.16. The method of claim 15 wherein the meat product is selected from thegroup consisting of a whole meat product and a processed meat product.17. The method of claim 16 wherein the processed meat product isselected from the group consisting of sausages, meat loaf, comminutedmeat product, ground meat, bacon, polony, salami, pate, an emulsifiedmeat product, and a restructured meat product.
 18. The method of claim15 wherein the dairy product is selected from the group consisting ofskimmed milk, whole milk, cream, a fermented milk product, cheese,yoghurt, butter, dairy spread, buttermilk, acidified milk drink, sourcream, whey based milk drink, ice cream, a flavoured milk drink, via,custard, other dessert products based on milk components, and mixturesthereof.